Evaluation of different approaches to identifying a higher throughput assay for time-dependent inhibition (TDI).

The availability of a reliable in vitro assay to evaluate time-dependent inhibition (TDI) of cytchrome P450 enzymes by novel compounds is essential for the identification of candidate medicines. We have evaluated three assay methods, making use of 59 marketed compounds and 28 novel GSK compounds. Recombinant bactosomes expressing the CYP3A4 isozyme were used with two fluorescence-based methods: a "Re-addition" assay and a "30 min" assay. The third method evaluated used pooled human liver microsomes (PHLM) with LC-MS/MS detection (the data for GSK compounds were evaluated in this study, whereas data for marketed drugs were reported recently). Our evaluation showed that the Re-addition method is comparable to the LC-MS/MS method in terms of predictivity and reproducibility. In conclusion, Re-addition method is inexpensive, and provides a simple assessment of the risk of TDI for novel compounds. This assay is particularly appropriate for use during the early stages of drug discovery.

Recognition of the kind of stress coping in patients of multiple sclerosis.

BACKGROUND: Investigations have shown that some factors like stress can increase the recurrence and severity of multiple sclerosis (MS). Considering the direct influences of depression and anxiety on our body immunity system, and also the relation between stress and factors, such as Insulin Growth Factor (IGF-1), involved in neurogenesis and myelin repairing, it is an essential issue to identify the most common method used in relieving stress by such patients. OBJECTIVE: To identify the type of common coping methods for stressful situation. MATERIALS AND METHODS: This case-control study was performed on 50 patients of both the genders with MS in Esfahan (Esfahan MS Association). The data were collected and then analyzed using analysis of variance (ANOVA) method with the help of SPSS software version 15. P value less than 0.05 was considered as statistically significant. RESULTS: In our study, coping method for stressful situation was significantly different in MS patients versus the healthy group (P=0.02). Descriptive indices showed that these patients use avoidant method more commonly than the control group (mean=45.01, SD=8.9 vs. mean=40.8, SD=11.8, respectively). CONCLUSION: Due to the different methods used by MS patients to cope with stressful situation in comparison with the healthy ones, more appropriate techniques can be introduced to modify them, and hence, less stress-induced side effects could be expected in this population.

Pharmacokinetics, metabolism and excretion of the glycine antagonist GV150526A in rat and dog.

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.

Identification of UDP-glucuronosyltransferases involved in the human hepatic metabolism of GV150526, a novel glycine antagonist.

The major metabolic pathway for elimination of GV150526 is by glucuronidation exerted by glucuronosyl transferases (UGTs). Potential exists for the modification of GV150526 pharmacokinetics by drugs capable of inhibiting the glucuronidation of GV150526. Using human liver microsomes, 44 compounds were screened for inhibition of GV150526 glucuronidation. These compounds were selected because they are extensively glucuronidated themselves or are used as concomitant medication in the treatment of acute stroke. For 11 compounds out of the 44, full inhibition kinetics were performed to determine their Ki-value and mechanism of inhibition. To predict possible in vivo drug-drug interactions, the theoretical percentage of inhibition (i) was determined, based on in vitro determined Ki-values, and the expected Cmax plasma levels of GV150526 and the inhibitor. Of the 11 compounds examined, only propofol had an i-value of 6.6; for all other compounds i-values were lower than 2.1. These results indicate that although in vitro inhibition is observed, the likelihood of in vivo drug-drug metabolic interactions occurring is low. The inhibition results suggest that in addition to UGT1A1, also UGT1A3, UGT1A8/9, and UGT2B4 are involved in the glucuronidation of GV150526. The involvement of UGT1A1 and UGT1A8/9 was confirmed from studies using cDNA expressed human UGT cell lines.

Human hepatic metabolism of a novel 2-carboxyindole glycine antagonist for stroke: in vitro-in vivo correlations.

1. The hepatic metabolism of 3-[-2(phenylcarbamoyl) ethenyl]-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, was investigated. 2. After a single intravenous administration of 800 mg GV150526 to healthy volunteers, six metabolites were observed. The major metabolites detected in human plasma have been shown by mass spectrometry to be glucoronides and one sulphate conjugate. 3. After incubation of GV150526 for 6 and 24 h with human liver slices, three glucuronide metabolites were observed. After incubation of GV150526 with pooled human liver microsomes, only one metabolite was observed, with the same molecular weight and HPLC retention time as the synthetic standard GV217053 (GV150526 hydroxylated on the para-position of the phenyl ring). 4. GV150526 hydroxylase enzyme kinetics--a step before sulphation--was determined using pooled human microsomes and was shown to be catalysed by cytochrome P4502C9. Glucuronidation kinetics towards GV150526 using microsomal preparations were also determined. Glucuronidation of GV150526 was observed with UGT1A1 cDNA-expressed protein, but not with UGT1A6. 5. The above enzyme kinetic data were used to calculate intrinsic clearance after scaling-up and hepatic clearance were calculated. Since GV150526 has a high plasma protein binding capacity, the effect of GV150526 binding to microsomal protein was determined. Thus, enzyme kinetic data were corrected, plotting the free (unbound) concentration of GV150526 versus enzymatic velocities: apparent Vmax did not alter significantly but apparent Km was approximately 10-fold lower. Correlation of these corrected enzyme kinetic data to predict clearance with in vivo clearance of GV150526 was good when both fu(plasma) and fu(microsomes) were included in the clearance calculations.

In vivo metabolism of a novel cholecystokinin B (CCK-B) antagonist in rat and dog plasma and rat faeces.

1. The in vivo metabolism of a novel CCK-B antagonist ((+)-N-[1-(adamantane-1-methyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H -1,5-benzodiazepin-3-yl]-N'-phenylurea, GV150013X) was investigated using rat and dog plasma (male and female) and rat faeces samples after administration of GV150013X. 2. Four monohydroxy and four dihydroxy metabolites of GV150013X were identified by comparison with authentic standards using hplc and results from previous in vitro studies. 3. In both rat and dog plasma, GV150013X was converted to one major and other minor metabolites. 4. Qualitatively there is no species or sex differences in the formation of metabolites except that minor metabolite M1 was not detected in dog plasma. 5. Traces of GV150013X and the major metabolite were seen in rat plasma sample 24 h after administration. 6. Hplc with UV and radiochemical detection was used to identify metabolites. Major, non-labelled GV150013X metabolites from rat faeces were collected for characterization by nmr.

Metabolism of a novel CCK-B antagonist in rat, dog and human liver microsomes.

1. The in vitro metabolism of a novel CCK-B antagonist ((+)-N-[1-adamantane-1-methyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H- 1, 5-benzodiazepin-3-yl]N'-phenyl-urea; GV150013X) was investigated using rat, dog and human liver microsomes. 2. Four monohydroxy and four dihydroxy metabolites of GV150013X in rat and man were identified by comparison with authentic standards using HPLC and mass spectrometry. 3. The dihydroxy metabolite M1 was not detected in dog liver microsomes mixtures. 4. The formation of dihydroxylated metabolites proceeds via monohydroxylated metabolites M5 and M8 and not directly from GV150013X. 5. A monohydroxy metabolite M5 was the major metabolite in rat and dog, with M5 and dihydroxy metabolites M2 and M3 major metabolites in man.

In vitro studies on the metabolic fate of mifentidine, a novel histamine H2-receptor antagonist.

The in vitro metabolism of mifentidine and several of its metabolites was studied using hepatic microsomes from seven animal species. The effects of potential enzyme inducers, inhibitors and activators were also studied. Mifentidine metabolites identified and characterised were: 4-imidazolylphenylamine (amine), 4-imidazolylphenyl-formamide (formamide), the urea derivative of mifentidine (urea) and the imidazole-hydroxylated derivative of the amine (i-OH-amine), along with three unidentified metabolites, M1, M2 and M3. Evidence for the presence of the amine, formamide, urea and i-OH-amine was obtained by comparison with authentic reference compounds: (i) HPLC retention times; (ii) UV spectra; and (iii) MS spectra of metabolites. The postulated intermediates are: carbinolimine (for formamide, amine, i-OH-amine and urea formation); formamide (for amine and i-OH-amine formation); amine (for i-OH-amine formation), and nitrone (for urea formation). One 'metabonate' of mifentidine was also identified, namely the nitro analogue of the amine. A possible prerequisite for the formation of this nitro is the corresponding hydroxylamine or nitroso compound. Cytochromes P450I and P450II were shown to be involved in the in vitro microsomal biotransformation of mifentidine, but the involvement of the flavin monooxygenase system was not proven.

In vitro metabolism of GV150013X by using liver microsomes and liver tissue slices from different species.

The metabolism of GV150013X was studied in vitro using washed liver microsomes and liver tissue slices from different species. This work was carried out in order to compare the metabolite profiles resulting from incubation of GV150013X with human, rat, dog and rabbit liver microsomes and those from rat, rabbit and human liver tissue slices. This compound was found to be converted to at least 8 metabolites by rat and human liver microsomes. In rabbit liver microsomes, the three metabolites M4, M7 and M8, and in dog liver microsomes metabolite M1, were not detected. The main metabolites, M2, M5 and M6, were present in human, rat and rabbit liver tissue slices, while the three metabolites M3, M4 and M8 and metabolite M1 were not detected in rabbit and rat liver tissue slices, respectively. In rat liver tissue slices, the major metabolites plus five minor metabolites, one sulphate conjugate of a monohydroxylated, three trihydroxylated and one dihydroxylated were identified based on HPLC retention time and thermospray-mass spectrometry data. Quantitative species differences among rat, dog, rabbit and human were observed, while qualitative differences only between rabbit and other species were detected.

Characterization of 2-amino-1-benzylbenzimidazole and its metabolites using tandem mass spectrometry.

We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocycle, 2-amino-1-benzylbenzimidazole (ABB). Three major metabolites were isolated and structurally characterized, using a combination of off-line HPLC, in conjunction with both electron ionization and fast atom bombardment ionization tandem mass spectrometry. ABB was shown to be debenzylated to afford 2-aminobenzimidazole (AB), as well as N- and C-oxidized to give 1-benzyl-N2-hydroxyaminobenzimidazole (BHB) and 2-amino-1-benzyl-hydroxybenzimidazole, respectively. The possible reasons for formation of the exocyclic hydroxylamine BHB are discussed. Furthermore, ABB is proposed as a suitable model compound for investigating parameters that control formation of toxic hydroxylamines derived from amino-azaheterocycles.

Characterization of N-benzylcarbazole and its metabolites from microsomal mixtures by tandem mass spectrometry.

The metabolism of N-benzylcarbazole (NBC) was studied in vitro using hamster hepatic microsomes to establish whether the corresponding amide is formed. This work was carried out in order to see if the extremely low pka characteristic of such a benzylic amine would allow the formation of the carbonyl derivative. No amide formation was observed. However, a number of metabolic products were detected using HPLC, including the oxidative debenzylation products, namely carbazole and benzaldehyde, together with 2 phenolic isomers of NBC. These products were tentatively characterized by their UV spectra using a rapiscan UV detector connected to HPLC equipment. The structural characterization of these 4 metabolites, together with unchanged substrate, was carried out using desorption electron impact tandem mass spectrometry (DEI-MS/MS) on a hybrid instrument with EBQ1Q2 configuration.

The influence of local antichlamydial antibody on the acquisition and persistence of human ocular chlamydial infection: IgG antibodies are not protective.

In order to study the effect of antichlamydial antibodies in ocular secretions on resistance to ocular chlamydial infection and clearance of this infection, we have performed linked longitudinal studies in a Gambian village in which trachoma is endemic. We have measured IgG and IgA antibody levels to a local serotype B isolate of Chlamydia trachomatis by amplified enzyme immunoassay, and chlamydial antigen levels in conjunctival swabs using a commercially available immunoassay which detects chlamydial glycolipid. Having previously demonstrated that sharing a bedroom with a case of active trachoma is a risk factor for acquisition of the disease, we have analyzed the effect of IgG and IgA antibody on the acquisition and persistence of clinical trachoma after controlling for age, sex, exposure to infection and for the presence of chlamydial antigen using a Poisson regression model. We have found that the presence of antichlamydial IgG in ocular secretions of disease-free subjects is associated with an increased incidence of trachoma. IgA antibody shows an opposite trend, but this is not statistically significant. One possible explanation of these findings is that antichlamydial IgG antibodies enhance the infectivity of C. trachomatis for the human eye; this could have major implications for the development of a chlamydial vaccine.

An evaluation of tandem mass spectrometry in drug metabolism studies.

The use of precursor ion and constant neutral loss scanning as a means of rapidly detecting drug metabolites is evaluated. Four clinically useful drugs, namely (i) cyclophosphamide, (ii) mifentidine, (iii) cimetropium bromide and (iv) haloperidol, were subjected to microsomal incubations to afford phase I metabolites. Aside from a minor clean-up procedure involving zinc sulfate precipitation of microsomal proteins and solid-phase extraction of metabolites using a Sep-pak C-18 cartridge, the mixtures were analysed directly by fast atom bombardment tandem mass spectrometry. It is demonstrated that such screening strategies are important in detecting novel metabolites. However, there are some problems associated with only using such methods, including (i) the possibility of not detecting metabolites that undergo unusual collision-induced dissociation fragmentation pathways, (ii) the non-detection of metabolites that have undergone metabolic change at unusual sites of reactivity, and (iii) production of artifacts derived from the parent drug by the primary ionization process. Examples are discussed that highlight both the strengths and weaknesses of such an approach.

Identification of metabolites of 4,4'-diaminodiphenylmethane (methylene dianiline) using liquid chromatographic and mass spectrometric techniques.

The in vitro metabolism of 4,4'-diaminodiphenylmethane (methylene dianiline, MDA) was investigated using rabbit liver microsomes. Minimal clean-up of the microsomal incubations was carried out using zinc sulphate precipitation followed by solid-phase extraction on Sep-Pak C18 cartridges. Three metabolites were detected in hepatic microsomal incubations, namely the azodiphenylmethane (azo) azoxydiphenylmethane (azoxy) and 4-nitroso-4'-aminodiphenylmethane (nitroso) compounds. The azo and azoxy metabolites were produced enzymatically whereas the nitroso compound may have been formed via a non-enzymatic process. Reversed-phase high-performance liquid chromatography-plasma spray mass spectrometry was used to initially detect these metabolites. Fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry were utilized to further structurally characterise these compounds. Comparison of mass spectral data obtained from synthesised standards with data obtained on the putative metabolites substantiated the characterisation of these compounds.

Bioanalytical applications of tandem mass spectrometry in the in vitro metabolism of the anticholinergic drug cimetropium bromide to detect differences in species metabolism.

1. In vitro metabolism of the anticholinergic drug, cimetropium bromide, was investigated using four different animal hepatic microsomal incubates derived from rat, hamster, guinea pig, and mouse livers. 2. Constant neutral loss (CNL) tandem mass spectrometry was used to detect the presence of the N-methylenecyclopropyl-scopine functionality by monitoring loss of 54 daltons (corresponding to loss of methylenecyclopropane) in microsomal incubates. 3. A CNL loss of 46 daltons was used to screen for the presence of ester hydrolysis products. 4. A comparison of the daughter ion spectra obtained on ions detected by CNL scanning, with daughter ion spectra of synthetic standards, determined the presence of ten metabolites of cimetropium bromide. 5. Hydroxylation of the aromatic ring in the ester side-chain was found to be the major metabolic pathway, and ester bond hydrolysis was a minor metabolic pathway. 6. N-Demethylation of the bridgehead nitrogen was observed only in rat and hamster incubates. 7. Using the method of CNL scanning it was possible to screen different animal microsomal incubates without resorting to any major purification procedures such as h.p.l.c. 8. This scanning method revealed differences between species in the metabolic pathways of cimetropium bromide.

Identification of cyclophosphamide-DNA adducts in rat embryos exposed in vitro to 4-hydroperoxycyclophosphamide.

Cyclophosphamide and other bifunctional alkylating agents are potent animal teratogens inducing a variety of malformations. Although cyclophosphamide-induced DNA damage is implicated as a primary mechanism underlying the teratogenesis initiated by cyclophosphamide, additional insights into the complex nature of the teratogenic process have been hampered by the inability to analyze the primary teratogenic lesions, i.e., cyclophosphamide-DNA adducts. Using tandem mass spectrometry, we show that the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (NOR-G) and bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-NOR-G) can be detected in the DNA of organogenesis-stage rat embryos after an in vitro exposure to an embryotoxic concentration of activated cyclophosphamide, i.e., 4-hydroperoxycyclophosphamide.

Rapid and efficient purification of cimetropium bromide and mifentidine drug metabolite mixtures derived from microsomal incubates for analysis by mass spectrometry.

A comparative study of the use of organic solvent extraction versus Sep-Pak C18 cartridges in the recovery and analysis of phase I (unconjugated) drug metabolites using mass spectrometry is presented. Standard mixtures of putative metabolites of the anticholinergic drug cimetropium bromide and the H2-antagonist mifentidine were purified from inactivated liver microsomal preparations using both methods, and subsequently the recovery of each compound was quantitated. In general, the percentage recovery and degree of purification were greater when using Sep-Pak C18 cartridges compared with organic solvent extraction. Even more efficient recovery was achieved when zinc sulphate precipitation of proteins in the liver microsomal mixtures was carried out prior to analysis. Also, the HPLC-grade solvents used in this study contained a variety of ultraviolet-inactive, hydrophobic components. This leads to problems of suppression in fast atom bombardment mass spectrometric analysis. Using Sep-Pak C18 cartridges directly prior to analysis by fast atom bombardment with single or tandem mass spectrometry leads to far superior mass spectral results compared with organic solvent extraction.

Simultaneous determination of carbamazepine and its major metabolites in human plasma and urine by HPLC.

A method is described for the analysis of carbamazepine and its major metabolites in biological fluids. The method uses a HPLC system based on a C18 Partisil column. The metabolites were extracted from chloroform in the presence of an internal standard. The method was specific, precise and reproducible.

Persisting inapparent chlamydial infection in a trachoma endemic community in The Gambia.

In a Gambian village with endemic trachoma, C. trachomatis was isolated from some 50% of children with active trachoma but from only 1% of adults with scarring sequelae. One possibility is that individuals with progressive scarring disease have clinically inapparent, non-productive infection which cannot be detected by cell culture. In whole village survey, 12% of 808 people carried ocular chlamydial antigen detected by EIA. Around 70% of antigen positive children were clinically active. By contrast, for adults age greater than 25 years, only 10% of antigen positives were clinically active. Antigen positive adults were twice as likely to develop scarring disease and longitudinal studies suggest that they are intermittently infectious. Implications for the pathology and prevention of trachoma are discussed.

A rationale for the non-mutagenicity of 2- and 3-aminobiphenyls.

Of the three isomeric forms of aminobiphenyl, only 4-aminobiphenyl is an established carcinogen while the 2-isomer is considered as a non-carcinogen and 3-aminobiphenyl is at best described as a weak carcinogen. In the present studies we investigated the mutagenicity of these three compounds, their N-hydroxy derivatives and their nitrosoderivatives in the Ames test using the Salmonella typhimurium strains TA98 and TA100. The studies were performed both in the absence and presence of an activation system derived from the liver of rats pretreated with Aroclor 1254. Of the three isomers only 4-aminobiphenyl exhibited mutagenicity and only in the presence of an activation system. N-Hydroxy-4-aminobiphenyl was a potent direct mutagen in both bacterial strains, N-hydroxy-2-aminobiphenyl was mutagenic in only TA100 while N-hydroxy-3-aminobiphenyl displayed mutagenicity in neither strain. Both 2- and 3-nitrosobiphenyls were direct mutagens in strain TA100. These findings suggest that the weak carcinogenicity of 3-aminobiphenyl may be attributed to the lack of genotoxicity of its N-hydroxyderivative, whereas in the case of 2-aminobiphenyl it may be due to the inability of the hepatic preparations to catalyse its N-hydroxylation, which is in agreement with published in vivo metabolic studies. It is interesting that of the three isomers only 2-aminobiphenyl is non-planar, forming a dihedral angle of 40 degrees, and this may preclude it from acting as a substrate of the P-450I family of haemoproteins, which selectively catalyses the N-hydroxylation of many aromatic amines including 4-aminobiphenyl.